Introduction: The amino acid transporter B0AT1 (Slc6a19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B0AT1 mediates the sodium-dependent uptake of all neutral amino acids. For surface expression and catalytical activity, B0AT1 requires coexpression of collectrin (TMEM27). In this study we established tools to identify and evaluate novel inhibitors of B0AT1.

Methods: A Chinese Hamster Ovary (CHO)-based cell line was generated, stably expressing collectrin and B0AT1. Using this cell line, a high-throughput screening assay was developed, which uses a fluorescent dye to detect depolarisation of the cell membrane during amino acid uptake via B0AT1. In parallel to these functional assays we ran a computational compound screen using AutoDock4 and a homology model of B0AT1 based on the high resolution structure of the highly homologous Drosophila dopamine transporter.

Results: We characterized a series of novel inhibitors of the B0AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC50 of 20±7μM. The compound was selective with regard to related transporters and blocked neutral amino acid uptake in inverted sections of mouse intestine.

Conclusion: The tools established in this study can be widely used to identify new transport inhibitors. Using these tools we were able to identify compounds that can be used to study epithelial transporters or be developed further through medicinal chemistry.